This project will explore the mechanism(s) responsible for decreased ristocetin-von Willebrand factor (VWF) interactions due to mutations and polymorphisms in the VWF A1 domain, the region of VWF most critical for interaction with platelet glycoprotein Ib (GPIb). The functional implications of these polymorphisms will be tested in the absence of ristocetin in order to determine their effect on VWF function. In addition, the role of factor VIII (FVIII) release following VWF-platelet binding will be investigated. The objective of this application is to determine the in vivo relevance of A1 domain polymorphisms that lead to decreased VWF activity on functional testing with ristocetin, to ensure that patients are not misdiagnosed with von Willebrand disease (VWD) on the basis of spurious laboratory testing results. Specific aim 1 will elicit the mechanism(s) behind the decrease in ristocetin-induced VWF-platelet interactions associated with A1 domain mutations through direct binding assays and surface plasmon resonance. Binding strength will be assessed with optical tweezers. Specific aim 2 will evaluate the effect(s) of key A1 domain mutations on platelet GPIb interactions independent of VWF as well as the mutations' effects on other aspects of VWF function. Both static and flow conditions will be assessed for VWF-collagen and VWF-platelet binding. Specific aim 3 will determine the impact of factor VIII (FVIII) release in clot formation following VWF-platelet interactions through measurement of FVIII activity and thrombin generation after ligand binding. Clot formation will be examined with in vivo thrombosis models. Bob Montgomery, MD, an experienced researcher in VWF biology, will serve as the primary mentor for this project, along with a committee of research scientists established for the purpose of enhancing the applicant's scientific knowledge and research career growth. This project will utilize the resources of the Medical College of Wisconsin and the Blood Research Institute of the Blood Center of Wisconsin to further the applicant's understanding of coagulation biology and scientific methods for examining coagulation factor function in vitro and in vivo. The applicant will also draw on the resources of a comprehensive clinic for patients with bleeding disorders for better understanding of the clinical behavior of VWF in patients. This environment provides a unique setting for training clinician scientists in the field of coagulation. The ultimate goal of this project is translation and application of these research findings to improve care for patients with VWD and other disorders of coagulation and to further an academic research career for the principal investigator.